Rational Design of the ß-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the ß-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the ß-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation.

Tyrosinase is a binuclear copper-containing metalloprotein that leads the fast and regio-selective o-hydroxylation of monophenols to o-diphenols. However, the subsequent second oxidation to produce o-quinones, i.e., melanin precursors, from the o-diphenols has restricted its use to the production of functional o-diphenol derivatives. Herein, we present a combined strategy for the effective inhibition of melanin formation in tyrosinase reaction, which allows the use of tyrosinase as a monophenol monooxygenase. The o-diphenolic products were protected from being oxidized in the tyrosinase reaction by borate ions and L-ascorbic acid (LAA). Borate-o-diphenol complexes were favorable formed at high pH and consequentially protected the o-diphenolic products from the catecholase activity of tyrosinase. LAA not only directly reduced the byproduct, o-quinones, into o-diphenols but also assisted the completion of the tyrosinase reaction cycle by removing a hydroxyl group attached to the copper metal cluster at the active site of the met-form tyrosinase. The regio-selective o-hydroxylation of 7,4'-dihydroxyisoflavone (daidzein) to produce 7,3',4'-trihydroxyisoflavone (3'-ODI) was successfully carried out by whole E. coli cell biotransformation with heterologously expressed tyrosinase from Bacillus megaterium. The yield of this o-hydroxylation of 5 mM daidzein in one-pot 400 mL reaction was ca. 100% in 90 min and the productivity was 16.3 mg 3'-ODI · L(-1) · h(-1) · DCW mg(-1), which is considerably higher than that of other monooxygenases. The method effectively abolished melanin synthesis, so that the o-diphenolic product remained stable without enzyme inactivation. Other monophenolic phytochemicals such as resveratrol and genistein could be subjected to the same strategy. After 1 h, 1 mM of genistein and resveratrol were both converted to orobol and piceatannol, respectively, with ca. 95% conversion yield. These results support the strong potential of tyrosinase as a monooxygenase for regio-selective o-hydroxylation of various monophenolic compounds.

Heterologous expression of tyrosinase (MelC2) from Streptomyces avermitilis MA4680 in E. coli and its application for ortho-hydroxylation of resveratrol to produce piceatannol.

Recombinant tyrosinase from Streptomyces avermitilis MA4680, MelC2 (gi:499291317), was heterologously expressed in Escherichia coli BL21 (DE3). The expression level of active MelC2 was increased by the codon-optimized MelC1 caddie protein (KP198295.1). By performing saturation mutagenesis of the Y91 residue of MelC1, it was found that aromatic residues such as Y, F, and W at the 91st position help produce a correctly folded conformation of MelC2. The recombinant MelC2 was utilized as a biocatalyst to convert trans-resveratrol into piceatannol. In order to improve the product yield through suppression of the formation of melanin, a by-product, an increase in the ratio of monooxygenation (k 1) to dioxygenation (k 2) of MelC2 is desirable. This was achieved by a combination of protein engineering and regeneration of NADH with glucose dehydrogenase (GDH). Saturation mutagenesis was performed at 15 residues within 8-Å radius from copper ions of MelC2. A total of 2760 mutants were examined (99.7 % probability for NNK codon) and I41Y, a mutant, was screened. The ratio of k 1 to k 2 of the mutant increased sevenfold on tyrosine and fivefold on resveratrol, when compared to wild-type MelC2. As a result, the overall product yield from 500 µM resveratrol in 50-mL reaction was 15.4 % (77.4 µM piceatannol), 1.7 times higher than wild type. When I41Y was incorporated with the NADH regeneration system, the total product yield was 58.0 %, an eightfold increase (290.2 µM of piceatannol).

Biophysical and in silico characterization of NrtA: a protein-based host for aqueous nitrate and nitrite recognition.

Nitrate and nitrite are key components of the global nitrogen cycle. As such, Nature has evolved proteins as biological supramolecular hosts for the recognition, translocation, and transformation of both nitrate and nitrite. To understand the supramolecular principles that govern these anion-protein interactions, here, we employ a hybrid biophysical and in silico approach to characterize the thermodynamic properties and protein dynamics of NrtA from the cyanobacterium Synechocystis sp. PCC 6803 for the recognition of nitrate and nitrite.

Controlling Nucleopeptide Hydrogel Self-Assembly and Formation for Cell-Culture Scaffold Applications.

Hydrogels made from self-assembling peptides have significant advantages in tissue engineering, namely a biocompatible nature and large molecular repertoire. Short peptides in particular allow for straightforward synthesis, self-assembly, and reproducibility. Applications are currently limited, however, due to potential toxicity of the chemical modifications that drive self-assembly and harsh gelation conditions. Peptides conjugated to nucleobases present one opportunity for a naturally derived species to minimize cytotoxicity. We have developed a hydrogel-formation environment for nucleopeptide gelation modulated entirely by biological buffers and salts. Self-assembly in this system is dependent on buffer and ion identity mediated by pKa and formulation in the former and by valency and ionicity in the latter. Solutions at physiological pH and osmolarity, and in turn compatible with cell culture, initiate hydrogel formation and analytical and computational methods are used to explore pH and salt effects at the molecular and structural level. The mechanism of nucleopeptide self-assembly enables tuning of mechanical properties through the addition of divalent cations and one order of magnitude increase in hydrogel storage modulus. The stability of these constructs therefore provides an opportunity for long-term cell culture, and we demonstrate survival and proliferation of fibroblasts on hydrogel surfaces. This novel, biological buffer-mediated gelation methodology expands opportunities for tissue engineering applications of short peptides and their derivatives.

The design and evolution of fluorescent protein-based sensors for monoatomic ions in biology.

Living cells rely on a finely tuned symphony of inorganic ion gradients composed of both cations and anions. This delicate balance is maintained by biological receptors all acting in concert to selectively recognize and position ions for homeostasis. These dynamic processes can be intercepted and visualized with optical microscopy at the organismal, tissue, cellular and subcellular levels using fluorescent protein-based biosensors. Since the first report of such tool for calcium (Ca2+) in 1997, outstanding biological questions and innovations in protein engineering along with associated fields have driven the development of new biosensors for Ca2+ and beyond. In this Review, we summarize a workflow that can be used to generate fluorescent protein-based biosensors to study monoatomic ions in biology. To showcase the scope of this approach, we highlight recent advances reported for Ca2+ biosensors and in detail discuss representative case studies of biosensors reported in the last four years for potassium (K+), magnesium (Mg2+), copper (Cu2+/+), lanthanide (Ln3+) and chloride (Cl-) ions.

Self-assembled nucleo-tripeptide hydrogels provide local and sustained doxorubicin release.

Self-assembled nucleo-peptide hydrogels have a nanofibril structure composed of noncovalent molecular interactions between peptide groups as well as π-π stacking and Watson-Crick interactions via complementary nucleobases. These hydrogels have specific benefits for biomedical applications due to their DNA-like interactions in addition to the well-known advantages of peptide biomaterials: biocompatibility, extracellular matrix (ECM)-like structure, and bottom-up design. Inspired by the nucleobase stacking structure, we hypothesized that nucleo-peptides would be able to deliver the DNA-intercalating chemotherapeutic, doxorubicin (Dox) in a sustained manner when delivered locally to a solid tumor. Ade-FFF nucleo-peptide hydrogels were able to load a high concentration of Dox (1 mM) and demonstrated continuous release under in vitro degradation conditions. We adopted an in vivo tumor-bearing mouse model to evaluate the delivery of Dox by Ade-FFF hydrogels. We found that Dox-containing hydrogels reduced tumor growth and resulted in greater apoptosis-mediated cell death in the tumor as evidenced by caspase-3 expression. Pharmacokinetics and biodistribution studies also supported the observation that Dox delivery by an Ade-FFF hydrogel improves sustained delivery in the local tumor site. This study demonstrates the potential of self-assembled nucleo-peptides in biomedical applications by using their distinctive DNA-like structure.

Design and Characterization of Nucleopeptides for Hydrogel Self-Assembly.

Self-assembling peptides can be used in a bottom-up approach to build hydrogels that are similar to the extracellular matrix at both structural and functional levels. In this study, a nucleo-tripeptide library was constructed to identify molecules that form hydrogels under physiological conditions. We used both experimental and computational approaches to study these self-assembled structures. Circular dichroism spectroscopy, transmission electron microscopy, and rheometry were utilized to support and supplement molecular dynamics simulations. Our data demonstrate that nucleo-tripeptides can form nanofibrous hydrogels through Watson-Crick base pairing and π-π stacking interactions. Self-assembly conditions are mediated by nucleo-tripeptide hydrophobicity and amphiphilicity and can therefore be regulated by a rational molecular design. We have found that structures derived from specific peptide and nucleobase conjugations form hydrogels under physiologic conditions, making them promising candidates for biomedical applications.

Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels.

Described herein is the design of a cell-adherent and degradable hydrogel. Our goal was to create a self-assembling, backbone ester-containing analogue of the cell adhesion motif, arginine-glycine-aspartic acid (RGD). Two depsipeptides containing Fmoc (N-(fluorenyl)-9-methoxycarbonyl), Fmoc-FR-Glc-D, and Fmoc-F-Glc-RGD (where "Glc" is glycolic acid) were designed based on the results of integrin-binding affinity and cell interaction analyses. Two candidate molecules were synthesized, and their gelation characteristics, degradation profiles, and ability to promote cell attachment were analyzed. We found that ester substitution within the RGD sequence significantly decreases the integrin-binding affinity and subsequent cell attachment, but when the ester moiety flanks the bioactive sequence, the molecule can maintain its integrin-binding function while permitting nonenzymatic hydrolytic degradation. A self-assembled Fmoc-F-Glc-RGD hydrogel showed steady, linear degradation over 60 days, and when mixed with Fmoc-diphenylalanine (Fmoc-FF) for improved mechanical stiffness, the depsipeptide gel exhibited improved cell attachment and viability. Though the currently designed depsipeptide has several inherent limitations, our results indicate the potential of depsipeptides as the basis for biologically functional and degradable self-assembling hydrogel materials.

Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily.

Herein, we report a two-step deglycosylation mediated by the oxidation of glycoside which is different from traditional glycoside hydrolase (GH) mechanism. Previously, we reported a novel flavin adenine dinucleotide (FAD)-dependent glycoside oxidoreductase (FAD-GO) having deglycosylation activity. Various features of the reaction of FAD-GO such as including mechanism and catalytic residue and substrate specificity were studied. In addition, classification of novel FAD-GO subfamily was attempted. Deglycosylation of glycoside was performed spontaneously via oxidation of 3-OH of glycone moiety by FAD-GO mediated oxidation reaction. His493 residue was identified as a catalytic residue for the oxidation step. Interestingly, this enzyme has broad glycone and aglycon specificities. For the classification of FAD-GO enzyme subfamily, putative FAD-GOs were screened based on the FAD-GO from Rhizobium sp. GIN611 (gi 365822256) using BLAST search. The homologs of R. sp. GIN611 included the putative FAD-GOs from Stenotrophomonas strains, Sphingobacterium strains, Agrobacterium tumefaciens str. C58, and etc. All the cloned FAD-GOs from the three strains catalyzed the deglycosylation via enzymatic oxidation. Based on their substrate specificities, deglycosylation and oxidation activities to various ginsenosides, the FAD-GO subfamily members can be utilized as novel biocatalysts for the production of various aglycones.